Reaction of PerR with Molecular Oxygen May Assist H2O2 Sensing inAnaerobesRamakrishnan Sethu,†,‡,§ Eric Goure,†,‡,§ Luca Signor,∥,⊥,# Christelle Caux-Thang,†,‡,§

Martin Clemancey,†,‡,§ Victor Duarte,*,†,‡,§ and Jean-Marc Latour*,†,‡,§

†Universite Grenoble Alpes, LCBM, F-38054 Grenoble, France‡CEA, DSV, BIG, LCBM, PMB, F-38054 Grenoble, France§CNRS UMR 5249, LCBM, F-38054 Grenoble, France∥Universite Grenoble Alpes, IBS, F-38044 Grenoble, France⊥CNRS, IBS, F-38044 Grenoble, France#CEA, IBS, F-38044 Grenoble, France

*S Supporting Information

ABSTRACT: PerR is the peroxide resistance regulator found in several pathogenic bacteria andgoverns their resistance to peroxide stress by inducing enzymes that destroy peroxides. However, ithas recently been implicated as a key component of the aerotolerance in several facultative or strictanaerobes, including the highly pathogenic Staphylococcus aureus. By combining 18O labelingstudies to ESI- and MALDI-TOF MS detection and EMSA experiments, we demonstrate that theactive form of PerR reacts with dioxygen, which leads ultimately to disruption of the PerR/DNAcomplex and is thus physiologically meaningful. Moreover, we show that the presence of O2 assistsPerR sensing of H2O2, another feature likely to be important for anaerobic organisms. Theseresults allow one to envisage different scenarios for the response of anaerobes to air exposure.

The generation of reactive oxygen species (ROS)deleterious to the cell is the price to pay for aerobic life.

These species which result from enzymatic partial reduction ofdioxygen include superoxide (O2

•−), hydrogen peroxide(H2O2), and the hydroxyl radical (HO•).1 Whereas it is notthe most aggressive, H2O2 must be tightly controlled because itis the immediate precursor of HO•, which attacks all cellcomponents. As a consequence, aerobic organisms haveevolved efficient enzymatic machineries able to cope withsuperoxide (superoxide dismutases, superoxide reductases) andperoxides (catalases, peroxidases) so as to maintain them at alow enough level compatible with cell survival.2 However, inthe case of a burst of ROS production (oxidative stress), thesedefenses are overwhelmed, and higher levels of defenseenzymes must be produced.3 Their synthesis is under controlof transcription factors which are highly sensitive to a specificROS and induce an adequate response to cope with theaggression. This is the case in particular for pathogenic bacteriafacing the ROS production by macrophages and neutrophils.2

The paradigm of this adaptive response is OxyR, a transcriptionfactor that activates ca. 20 genes involved in H2O2

detoxication.4 OxyR operates thanks to a pair of activecysteines which forms a disulfide by reaction with low levelsof H2O2, the conformational change associated with theformation of this disulfide being the trigger of the response

to initiate gene expression.4 OxyR is the peroxide sensor ofGram-negative bacteria such as Escherichia coli.The functional equivalent of OxyR in Gram-positive bacteria

is the metalloregulator PerR discovered by Helmann’s group inBacillus subtilis.5 PerR was subsequently found in many otherorganisms,6−11 including virulent pathogens such as Staph-ylococcus aureus,12 Borrelia borgdurfori,11 and the Gram-negativeCampylobacter jejuni.6 PerR belongs to the Fur family ofmetalloregulators,13 and its structure is well-known thanks tothe X-ray crystallographic characterization of several forms ofthe Bs protein.14,15 The active form of PerRBS (noted PerR:Zn−Fe as opposed to PerR:Zn-apo, which lacks the regulatorymetal) is a dimer that contains two metal sites: a structural sitecomprising a Zn2+ ion tetrahedrally bound to four cysteinates14

and a regulatory site which binds either Fe2+ or Mn2+ throughthree histidines (His37, His91, and His93 according to Bs PerRnumbering) and two aspartates (Asp85 and Asp104).15 Thedifferent roles of the two metals have been elucidated: the Zn2+

ion stabilizes the homodimer form of the protein,14 and bindingthe second metal in the regulatory site is necessary for theprotein to adopt the caliper-like shape requested for binding

Received: December 19, 2015Accepted: March 10, 2016Published: March 10, 2016

Articles

pubs.acs.org/acschemicalbiology

© 2016 American Chemical Society 1438 DOI: 10.1021/acschembio.5b01054ACS Chem. Biol. 2016, 11, 1438−1444

DNA.15 PerR controls a set of genes involved in peroxideresponse, and consistently, its regulon comprises enzymesinvolved in peroxide destruction, among which are the hemecatalase KatA and the alkylhydroperoxide reductase AhpC.16

PerR senses H2O2 by a mechanism17 which departs from theclassical cysteine oxidation of OxyR,4 the organic hydro-peroxide sensor OhrR,18,19 and many thiol peroxidases.20

Indeed, reaction of H2O2 with the regulatory Fe2+ ion causesthe oxygenation of a histidine ligand, either His37 or His91, toan oxo-histidine.17,21 This in turn leads to disruption of themetal binding site and eventually a loss of the caliper-likestructure, resulting in dissociation from DNA.17,21

Although PerR is described as a peroxide sensor in allorganisms in which it is present, it has also been implicated inaerobic stress of several anaerobes. A very recent report by VanVliet et al. indicated that PerR plays an important role in theaerobic survival of the foodborne bacterial pathogenCampylobacter jejuni, which is a microaerophile.22 This findingis reminiscent of the observation that PerR regulon is activatedby low-oxygen exposure in the anaerobe Desulfovibrio vulgarisHildenborough.23 A clear link between PerR and aerotolerancewas established for the strict anaerobe Clostridium acetobutyli-cum by Bahl et al.24 By comparing the wild type strain with aperR-deleted strain, these authors were able to propose thatPerR acts as an oxygen-dependent switch to allow a temporaryresponse to oxygen exposure. It was hypothesized that thisresponse was initiated by a low level of H2O2 acting as a generalsignal of aeration. Recently, Helmann et al. reported similarlythat the human anaerobe pathogen Staphylococcus aureusexhibits a hypersensitivity of PerR to Fe-mediated oxidationunder aerobic conditions and that it is due to the ability ofPerRSA to respond to very low levels of H2O2 encounteredduring aerobic growth.25 Hence, it appears that activation ofPerR peroxide regulon is involved in aerotolerance in manyanaerobes. either strict or facultative. These results prompted usto report our findings that the active form PerR:Zn−Fe ofPerRBS is able to react with dioxygen as it does with H2O2,leading to histidine oxygenation and disruption of the PerR−DNA complex. In addition, we observed that the presence of aircontributes substantially to PerR oxidation by H2O2. Bothobservations may shed new light on PerR physiological activity.

■ RESULTS AND DISCUSSIONPerR Oxidation during Growth. As reported by Lee and

Helmann,17 PerR is highly susceptible to oxidation duringgrowth and purification. For example, PerR purified underredox cycling conditions, in the presence of a thiol-reducingagent in EDTA lacking buffers, was found to be oxidized atmultiple sites. This multiple oxidation is significantly reduced inthe absence of DTT during sample preparation. Under theseconditions, the oxidation of PerR typically leads to a proteinhaving partly incorporated a single oxygen atom into eitherH37 or H91 residue. We observed that variable amounts (ca.30−60%) of singly oxidized PerR are obtained depending onthe preparation. By contrast, when the cells are grown inminimal medium supplemented with desferroxamin, a well-known ferric chelator, PerR:Zn-apo, could be isolatedreproducibly with very low protein oxidation levels (<10%).PerR:Zn-apo could then be metalated by 1 h incubation understrict anaerobic conditions (inert atmosphere chamber) withone equivalent of Fe2+, leading to the active form PerR:Zn−Fe.The Reaction of PerR Active Form with Molecular

Oxygen Leads to Oxidation of Histidines 37 and 91. The

metalated PerR:Zn−Fe protein kept under anaerobic con-ditions was incubated with an aerated buffer solution in a sealedmicrotube. The samples were then analyzed by ESI-TOF MS.Figure 1B shows that the peak at m/z 16292 Da corresponding

to unoxidized PerR is flanked by a smaller one at m/z 16308Da, the mass increase of +16 Da corresponding to the fixationof one oxygen atom. This indicated that PerR had beenoxidized by dioxygen at ca. 28% as judged by the relativeheights of the two peaks. The origin of the latter atom wastracked by using 18O labeled dioxygen. In this case, ESI-TOFMS analysis of the oxidized protein revealed that the peak ofthe oxidized protein has moved to m/z 16 310 Da (Figure 1C).The corresponding mass increase of +18 Da shows that theextra oxygen comes from O2. In these experiments, the possibleintermediate formation of H2O2 was assessed by adding catalasein the buffer solution as suggested previously.17 Figure 1Dshows that the peak at +16 Da is slightly decreased whencompared to the corresponding peak shown in Figure 1B. Theamounts of the singly oxidized proteins are 21% and 28%,respectively. The fact that catalase partially suppresses theaerobic oxidation of PerR suggests that, under these conditions,low amounts of H2O2 are generated and can lead to PerRoxygenation. This result is in agreement with previous workreported by Lee and Helmann17 who observed that catalasetotally inhibited PerR oxidation. These authors proposed thatone plausible way to form low levels of H2O2 is to generatesuperoxide anions by reaction of Fe2+ with O2; H2O2 is in turngenerated by dismutation of the superoxide anions. Interest-ingly, in the present study, catalase does not completelysuppress PerR oxidation, thus suggesting that a directinteraction of PerR-Fe with O2 may be operative and lead tohistidine oxygenation.MALDI-TOF mass spectrometry was used to identify the

sites of PerR oxidation in the presence of dioxygen. Figure 2A(H37) and B (H91) show that the additional oxygen atom isincorporated into the tryptic peptides that contain H91 andH37 residues, respectively. The levels of oxygenation for theH37 and H91 containing peptides are approximately 15% and5%, respectively, in satisfactory agreement with ESI-TOF MSresults. It is worth noting that a similar preference for His37oxygenation was found for PerR in vivo oxidation by H2O2.

17,21

Figure 1. ESI-TOF MS analysis of oxidation of PerR:Zn−Fe bymolecular oxygen. Top row: as-isolated protein (A), after oxidation by16O2 (B), by

18O2 (C), and by 16O2 in the presence of catalase (D).

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Anaerobic Oxidation of PerR by Hydrogen PeroxideLeads to Similar Levels of Oxidation of Histidines 37and 91. Before assessing the effect of dioxygen on thehydrogen peroxide oxidation of PerR, we established thereactivity of PerR with hydrogen peroxide on a quantitativebasis. The active form of the protein (PerR:Zn−Fe) wasincubated for 30 min with increasing concentrations, rangingfrom 10 to 50 μM (0.2 to 1 equiv per Fe) of hydrogen peroxideprepared in a deaerated buffer. The protein samples wereanalyzed by ESI-TOF MS to assess their level of oxidation. Thetop and middle rows of Figure 3 present the spectra obtainedfor PerR oxidation with H2O2 under anaerobic conditions. Asexpected, the increase in hydrogen peroxide concentrationleads to the increased formation of oxidized PerR that has

incorporated one oxygen atom (peak at m/z 16308 ± 1 Da).The extent of mono-oxidized PerR reaches ca. 35% in thepresence of 50 μM (1 equiv) H2O2. A minor peak at +32 Da,corresponding to the protein that contains two additionaloxygen atoms, starts appearing at 30 μM H2O2. Exposure ofPerR:Zn−Fe to higher concentrations of hydrogen peroxideleads to the oxidation of the protein at multiple sites. Typically,in the presence of 250 μM H2O2 (5 equiv to PerR:Zn−Fe), themajor peaks were found at +32 and +48 Da (Figure 3, bottomleft).MALDI-TOF MS analyses were performed on three trypsin

digested samples. Figure 2C (H37) and D (H91) illustrate thetwo H37 and H91 containing peptides after anaerobicoxygenation by 1 equiv of H2O2. Similar oxygenation levelsare observed for H37 (18%) and H91 (14%). When excessH2O2 is used under the same conditions, both peptides areagain oxidized to similar extents: at 2 equiv of H2O2, 23% vs22% for H37 and H91, respectively (SI, Figure S1), and at 5equiv of H2O2, 78% vs 77% for H37 and H91, respectively(Figure 2E (H37) and F (H91)). Moreover, as shown in thelatter example, their predominant simultaneous oxidationsuggests that, in the presence of a large excess of H2O2, bothH37 and H91 residues can be simultaneously oxidized.

Aerobic Conditions Assist PerR Sensing of HydrogenPeroxide. To evaluate the effect of dioxygen on the reactivityof PerR with hydrogen peroxide, the PerR:Zn−Fe protein wasincubated for 30 min with increasing concentrations ofhydrogen peroxide prepared in an aerated buffer. The proteinsamples were analyzed by ESI-TOF MS to assess their level ofoxidation (Figure 4). In contrast to the results observed whenH2O2 was prepared anaerobically, the peak of the mono-oxidized protein is the most intense, and the progression of theoxidation with increasing H2O2 is apparent from the decrease ofthe unoxidized protein (peak at 16292 Da). This illustrates thatthe extent of mono-oxidized protein is significantly higher whenH2O2 is prepared in an aerated solution. For example, aconcentration as low as 10 μM of aerated H2O2 leads to 55% ofthe +16 Da peak, a value which is approximately 4 times higherthan the one observed when a deaerated H2O2 solution wasadded. When PerR:Zn−Fe is reacted with a stoichiometricconcentration of aerated H2O2, the amount of the singly

Figure 2. MALDI-TOF MS analysis of peptides digested with trypsin (A−F) or LysC (G,H) showing the oxygenation of histidine 37 (A, C, E, G;m/z 2401 or 3538) and histidine 91 (B, D, F, H; m/z 2084 or 3163) after oxidation of PerR:Zn−Fe by molecular oxygen (A, B), by 1 equiv of H2O2in anaerobic buffer (C, D), by 5 equiv of H2O2 (E, F), and by 1 equiv of H2O2 in aerobic buffer (G, H).

Figure 3. Top and medium rows: ESI-TOF MS spectra of PerR:Zn−Fe after incubation with increasing amounts of H2O2 (0, 0.2, 0.4, 0.6,0.8, and 1 equiv). Bottom row: ESI-TOF MS spectra of PerR:Zn−Feafter incubation with 5 equiv of H2O2 (left).

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oxidized protein represents approximately 80%, whereas it wasonly 35% in absence of air (see above).MALDI-TOF MS analysis of the LysC digested PerR sample

in the presence of 1 eq of aerated H2O2 is shown in Figures 2G(H37) and 2H (H91). The MALDI-TOF MS spectra indicatethat both H37 and H91 containing peptides are oxidized. It hasto be noted that under these conditions the H37 containingpeptide is preferentially oxidized, as was observed when O2 wasthe oxidizing agent. The percentages of oxygenation are 55 and25 for the H37 and H91 containing peptides, respectively.These results are consistent with the previously reported massspectral data for the in vivo oxidation of PerR.17,21

Figure 5 shows the percentage of the singly oxidized PerRafter incubation with increasing concentrations of H2O2 in both

deaerated and aerated buffers. This graph reveals twointeresting features. First, in both cases a linear dependenceof the oxygenation extent on H2O2 concentration is observed.The linear regressions gave slopes of 49 and 61% per H2O2equivalent for deaerated and aerated H2O2, respectively. Thisdifference is marginally significant. Second, comparison of thedata obtained for the aerobic vs anaerobic conditions showsthat aeration contributes an almost constant (ca. 35%)oxygenation. Overall, these graphs illustrate that (i) at lowH2O2 concentration, dioxygen contribution may dominate and(ii) total and specific oxygenation of PerR in anaerobicconditions would require high concentrations of H2O2, whichin our experiments lead to multiple oxidation and nonspecific

degradation of the protein, whereas aerobic conditions warrantthat it can be reached at a H2O2/Fe ratio close tostoichiometry.Electrophoretic Mobility Shift Assay (EMSA) experiments

were performed to correlate the efficiency of the oxidativetreatment of PerR:Zn−Fe and the resulting DNA bindingcapacity of the oxidized protein. For these EMSA experiments,PerR:Zn-apo was first incubated with 1 equiv of Fe2+ and thentreated with a stoichiometric amount of H2O2 either deaeratedor not prior to gel analyses (Figure 6). Before H2O2 treatment,

the affinity of PerR for DNA is KD ≈ 1 nM (Figure 6 left). Afteranaerobic incubation with H2O2, the affinity is reduced 4 times,KD ≈ 4 nM (Figure 6 middle); in other words 4 times moreprotein is needed to bind the same amount of DNA. WhenH2O2 treatment is performed in the presence of air, the affinityis further reduced more than 5 times, KD > 20 nM (Figure 6right). Interestingly, the latter DNA binding pattern is highlysimilar to the one observed previously for the in vivo oxidationof PerR.21 These results clearly show that the presence of airsignificantly increases PerR oxidation by H2O2.

Physiological Significance of PerR Interaction withDioxygen. The role and action of PerRBS in peroxide sensingis well established: the reaction of H2O2 with the active formPerR:Zn−Fe causes the oxygenation of histidines 37 or 91 andthe disruption of the PerR/DNA complex, thus opening theway to expressing the various proteins constituting PerRBSregulon. As mentioned above, KatA and AhpC are central tothe peroxide response, but the regulon comprises also a set ofproteins dealing with iron homeostasis: a dps-like protein(MrgA), the ferric uptake regulator (Fur), an Fe(II) effluxprotein (PfeT, formerly ZosA),26 and proteins involved inheme biosynthesis.27 Indeed, capitalizing on their originalobservation that the Mn active form of PerR (PerR:Zn−Mn)acts as a repressor of the peroxide regulon,28 Helmannhighlighted recently that PerR plays an unanticipated role insensing the cellular Mn/Fe ratio,29 which has emerged recentlyas of the utmost importance to fight oxidative stress.30 Henceperoxide sensing is only a part of the physiological role of PerRin oxidative stress.Contrary to initial expectations, anaerobes also possess

enzymatic defenses against peroxide stress which enable themto survive temporary exposure to aerobic conditions.31 Asoutlined above, the role of PerR in the aerotolerance ofanaerobic organisms has been evidenced in several instan-ces,22−25 and the current hypothesis for its involvement impliesthe prerequisite formation of hydrogen peroxide by thenumerous enzymes maintaining these organisms in a reducingenvironment. Our results demonstrate that, beforehand, H2O2generation is not mandatory. First, PerR active form PerR:Zn−Fe is able to react with dioxygen in a manner that is

Figure 4. ESI-TOF MS spectra of PerR:Zn−Fe after incubation withincreasing amounts of H2O2 (0, 0.2, 0.4, 0.6, 0.8, and 1 equiv preparedin an aerated buffer).

Figure 5. Left: Percentage of monooxidized protein (+16 Da) aftertreatment of PerR:Zn−Fe by H2O2 in the presence (red bar) vsabsence (blue bar) of air (data from Figures 3 and 4). Right: Linearregression of these anaerobic (blue) and aerobic (red) data.

Figure 6. EMSA experiments comparing the binding of DNA to PerRbefore (left), after anaerobic (middle), and after aerobic (right) H2O2treatment.

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physiologically meaningful, i.e., oxygenation of histidines 37and 91 (Figure 1) and dissociation from DNA (Figure 6).Second, the presence of catalase has only a minor inhibitoryeffect (ca. 7%; Figure 1D), which rules out the requisiteintermediacy of H2O2 in the process. In their seminal paper,Lee and Helmann observed total inhibition of PerR oxidationby catalase.17 However, they used a 10-fold excess of Fe2+ withrespect to PerR, and in these conditions it is likely that thegreatest part of O2 was reduced to H2O2 by “free” ferrous ionsavailable and reacted with PerR:Zn−Fe. By consuming thusformed H2O2, catalase can also deprive the medium from O2,resulting in partial inhibition. In such a way, catalase could havealso partially prevented air-oxidation of PerR:Zn,Fe in theEMSA experiments performed by Lee and Helmann. In our invitro experiments, we took much care that all Fe ions be boundto the PerR active site by working under stoichiometric orsubstoichiometric Fe/PerR conditions. Under these conditions,interaction of O2 with the PerR:Zn−Fe active form ismandatory. Release of superoxide as required by Fentonreaction would generate an inactive ferric- or apo-PerR form.Finally, MALDI-TOF analyses of H37- and H91-containingpeptides after aerobic oxygenation reveal a preference for H37oxygenation, whereas in the absence of O2 both histidines areoxidized to similar extents. This result strongly suggests distinctmechanisms for O2 and H2O2 oxygenations. These mechanisticdifferences are highlighted in Scheme 1.

PerR active form PerR:Zn−Fe can interact with H2O2 asproposed by Lee and Helmann17 to generate a hydroxyl radical(Fenton reaction, path a) or, alternatively, to produce a high-valent FeIV ion (path b). As a matter of fact, it was recentlyshown that, at near-neutral pH, the alternate pathway maydominate the usual hydroxyl radical route.32 The differencebetween the two pathways originates in the homolytic (path a)vs heterolytic (path b) cleavage of the O−O bond of the initialperoxido intermediate. On the other hand, reaction of ferrousproteins with dioxygen has been shown to give a superoxoiron-(III) species (path c) which is able to attack substrates. O−Obond cleavage of the resulting peroxo derivative gives theoxygenated product.33 Such a mechanism can be posited forPerR interaction with dioxygen.All of these observations lead to consideration of the

possibility that PerR may behave as an oxygen sensor. In thisrespect, PerR histidine oxygenation to 2-oxohistidine bears astrong resemblance to proline oxygenation by prolyl hydrox-ylases of the Hypoxia Inducible Factor,34 which coordinatesmammalian responses to hypoxia. The quantitative data of

Figure 5 suggest that PerR interaction with dioxygen may havetwo complementary effects: early detection of aeration andassistance of the peroxide response. Whereas the latter effectwould be beneficial to all organisms, the former might beessential for strict anaerobes. The observation that PerRSA is farmore sensitive to H2O2 than PerRBS may illustrate such effects.These in vitro molecular observations must be complementedby in vivo quantitative experiments to investigate how the twoeffects are interconnected. To summarize, our uncovering ofthe oxygen sensitivity of PerR raises the question of its potentialrole as an oxygen sensor in anaerobes. In addition, it brings newclues on the mechanisms of aerotolerance.

■ METHODSProduction, Expression Conditions, and Purification of PerR.

The B. subtilis perR gene was inserted between NdeI and XhoIrestriction sites of pET-30c vector (Novagen). The plasmid wasintroduced into E. coli BL21(DE3) competent cells (Stratagene), andthe cells were grown aerobically at 37 °C in 1 L of Na2HPO4/KH2PO4buffered M9 medium containing 9 mM NaCl, 19 mM NH4Cl, 1 mMMgSO4, 100 μM CaCl2, 100 μMMnCl2, 50 μM ZnSO4, 4 g/L glucose,1 mg/L pyridoxine-HCl, 1 mg/L folic acid, 1 mg/L choline chloride, 1mg/L niacinamide, 1 mg/L D-biotin, 1 mg/L D-pantothenic acid, 0.1mg/L (−)-riboflavin, 5 mg/L thiamine-HCl, and 5 μM desferriox-amine. The cells were grown with 50 μg/mL kanamycin until 0.6 ODat 600 nm. Production of PerR-WT was induced by adding 1 mMIPTG, followed by 3 h of incubation at 37 °C. Cultures were thencentrifuged at 7000g for 10 min. From this point, all steps wereperformed at 4 °C. Cells were resuspended in 30 mL of 100 mM Tris-HCl at a pH of 8, 20 mM NaCl, and 10 mM EDTA (buffer A) prior tosonication. The cell lysates were then centrifuged for 30 min at25 000g. The resulting supernatants were directly loaded onto a Hiload16/10 Q sepharose High Performance column (GE Healthcare).Proteins were eluted with a linear gradient of 0.02−1 M NaCl in bufferA. The fractions containing PerR-WT were pooled and concentratedby ammonium sulfate precipitation (80%) at 4 °C overnight. Aftercentrifugation at 25 000g for 45 min, the pellets were resuspended in a4 mL solution of chelex-100 (Bio-Rad) treated with 100 mM Tris-HClat a pH of 8 and 250 mM NaCl and subjected to gel filtration using aSuperdex-75 column (GE Healthcare). The fractions containing thedimeric protein were then collected and stored at −80 °C. Theconcentrations of PerR-WT were calculated using an extinctioncoefficient of 9020 M−1 cm−1 at 277 nm.

Reactivity of PerR-Fe2+ with H2O2/O2. First, 50 μM PerR wasincubated with 1 equiv of Fe(NH4)2(SO4)2·6H2O under anaerobicconditions in 50 μL of 100 mM Tris-HCl at a pH of 8 and 250 mMNaCl for 1 h at 25 °C. The samples were then treated anaerobicallywith increasing concentrations of H2O2 for 30 min at 25 °C. The 10 Mstock solution of H2O2 was diluted with either a degassed or aeratedbuffered solution. For the experiments with 16O2, PerR-Fe wasincubated with an aerated buffer solution; a concentration of 250 μMdissolved dioxygen was considered. When using 18O2, 5 mL of thebuffer solution was first deaerated prior to bubbling 18O2 into thesolution. The different buffered solutions were kept in sealed vialsinside the glovebox. The required volumes of these solutions weremixed with the PerR-Fe protein by using a syringe. For the experimentwith catalase (Sigma-Alridch, bovine liver, 25 000 U/mL), a finalconcentration of 5 U/μL was used. This enzyme was incubated withPerR-Fe before adding the aerated buffer solution. After the additionof 500 μM EDTA, the samples were recovered in 70 μL of 10 mMTris-HCl at a pH of 8 and 25 mM NaCl by using Micro Bio-SpinChromatography Columns (Bio-Rad) for buffer exchange. Theconcentrations of the Fe2+/H2O2-treated PerR were determined byusing an extinction coefficient of 9020 M−1 cm−1 at 277 nm. Thesamples were then further analyzed by mass spectrometry or submittedto EMSA experiments by using the protocol described below.

Mass Spectrometry Analysis of the PerR. Liquid Chromatog-raphy Electrospray Ionization Mass Spectrometry (LC/ESI-MS) was

Scheme 1. Possible Mechanistic Pathways for PerROxidation

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applied to monitor oxidation on the intact protein by using a 6210LC/ESI-TOF mass spectrometer interfaced with an HPLC pumpsystem (Agilent Technologies). MS acquisition was carried out in thepositive ion mode with spectra in the profile mode. The MSinstrument was operated with the following experimental settings: ESIsource temperature was set at 300 °C; nitrogen was used as a dryinggas (7 L/min) and as nebulizer gas (10 psi); the capillary needlevoltage was set at 4000 V. The spectra acquisition rate was 1.03spectra/s. All solvents used were HPLC grade (Chromasolv, Sigma-Aldrich); trifluoroacetic acid (TFA) was from Acros Organics (puriss.,p.a.). Solvent A was 0.03% TFA in water; solvent B was 95%acetonitrile/5% water/0.03% TFA. The MS spectra were acquired andthe data processed with MassHunter workstation software (v. B.02.00,Agilent Technologies) and with GPMAW software (v. 7.00b2,Lighthouse Data, Denmark). The mass spectrometer was calibratedin the m/z 300−3000 range with standard calibrants (ESI-L, lowconcentration tuning mix, Agilent Technologies) before each series ofmeasurements. Just before analysis, the protein samples were dilutedunder acidic denaturing conditions to a final concentration of 5 μMwith solution A (0.03% TFA in water). Samples were thermostated at10 °C in the autosampler, and the analysis was run by injecting 4 μL ofeach sample. They were first trapped and desalted on a reverse phase-C8 cartridge (Zorbax 300SB-C8, 5 μm, 300 μm ID × 5 mm, AgilentTechnologies) for 3 min at a flow rate of 50 μL/min with 100%solvent A and then eluted with 70% solvent B at flow rate of 50 μL/min for MS detection. The RP-C8 cartridge was then re-equilibratedfor 4 min with 100% solvent A at a flow rate of 50 μL/min. Massspectra were recorded in the 300−3000 mass-to-charge (m/z) range.Peptide oxidation analyses were mapped by endoproteinase trypsin

or LysC digestion followed by MALDI-TOF mass spectrometryanalysis using an Autoflex mass spectrometer (Bruker Daltonics,Bremen, Germany) in the reflectron positive ion mode detection. Forprotein digestion, 20 μL of 12.5 μM PerR in 10 mM Tris-HCl at a pHof 8 were incubated with 240 ng of trypsin (Proteomics grade, Sigma-Aldrich) or 100 ng of endoproteinase LysC (Roche Applied Science)at 37 °C for 16 h. Digests were then diluted to 0.5 μM with the matrixsolvent (water/acetonitrile/formic acid, 50:50:0.2). A total of 0.5 μL ofthe sample was then mixed with an equal volume of matrix (α-cyano-4-hydroxy cinnamic acid, 10 mg mL−1 in water/acetonitrile/trifluoro-acetic acid:50/50/0.1) on the target plate and allowed to air-dry.For the calculation of the relative percentage of PerR oxidation, the

peak height values of the ESI-TOF MS deconvoluted spectra weretaken in consideration. For the MALDI-TOF MS analyses on peptides,the peak height values corresponding to the first peak (monoisotopicpeak) for each species were considered.DNA Binding of PerR. First, 50 μM PerR was incubated with 1

equiv of Fe(NH4)2(SO4)2·6H2O under anaerobic conditions in 50 μLof 100 mM Tris-HCl at a pH of 8 and 250 mM NaCl for 1 h at 25 °C.The samples were then treated either anaerobically or aerobically with50 μM H2O2 for 30 min at 25 °C. The resulting samples wereincubated with 1 mM EDTA for 15 min and then purified by usingBio-Gel P6 columns (Micro Bio-Spin chromatography columns, Bio-Rad). PerR samples were diluted to the desired concentration andthen incubated with 200 pM of a 33 bp labeled DNA duplex, in thepresence of 100 μM MnCl2. EMSA experiments were performed withChelex-100 treated buffers (storage, binding, and electrophoresisbuffers). One strand of the 33 bp oligonucleotide duplex (Eurofins)containing the PerR box from the mrgA promoter was initially 32P-5′-end labeled using [γ-32P]-ATP and T4-PNK (Fermentas). Annealingwith a stoichiometric amount of the complementary strand wasperformed in 20 mM Bis-Tris borate at a pH of 8, 50 mM KCl, and0.1% triton X-100, by heating at 90 °C for 10 min followed by slowcooling to 25 °C. To ensure the duplex formation, the labeled singlestrand and the duplex were loaded on a native gel. Binding reactionswere carried out for 15 min at 25 °C in 10 μL of 20 mM Bis-Trisborate at a pH of 8, 50 mM KCl, 100 μM MnCl2, 10 μg/mL dIdCDNA, 0.1% triton X-100, and 2.5% glycerol that contained the DNAduplex and the indicated protein concentrations. The samples werethen subjected to electrophoresis on a 15% polyacrylamide gel for 75min at 200 V, in 100 mM Bis-Tris borate at a pH of 8 and 100 μM

MnCl2. The gel was then exposed on a PhosphorImaging screen (Bio-Rad), visualized using a Molecular Imager FX (Bio-Rad), and analyzedusing Quantity One software (Bio-Rad).

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acschem-bio.5b01054.

Figure S1. MALDI-TOF MS (PDF)

■ AUTHOR INFORMATIONCorresponding Authors*E-mail: victor.duarte@cea.fr.*E-mail: jean-marc.latour@cea.fr.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSJ.-M.L. and V.D. acknowledge the financial support of IFCPAR(Project No. IFC/4109-1) and Labex ARCANE (ANR-11-LABX-0003-01). This work used the platforms of the GrenobleInstruct Centre (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL)with support from FRISBI (ANR-10-INSB-05-02) and GRAL(ANR-10-LABX-49-01) within the Grenoble Partnership forStructural Biology (PSB).

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